Journal of the American College of Cardiology
© 2002 by the American College of Cardiology Foundation
Published by Elsevier Science Inc.
Vol. 40, No. 5, 2002
ISSN 0735-1097/02/$22.00
PII S0735-1097(02)02042-9
Deoxyribonucleic Acid Damage
in Human Lymphocytes After
Percutaneous Transluminal Coronary Angioplasty
Maria Grazia Andreassi, BSC, Nicoletta Botto, BSC, Antonio Rizza, MD, Maria Giovanna Colombo, BSC,
Cataldo Palmieri, MD, Sergio Berti, MD, Samantha Manfredi, BSC, Serena Masetti, BSC,
Aldo Clerico, MD, Andrea Biagini, MD
Massa, Italy
We investigated the presence of oxidative deoxyribonucleic acid (DNA) damage in the
peripheral lymphocytes of patients undergoing percutaneous transluminal coronary angioplasty (PTCA) by using the micronucleus test and comet assay, which are sensitive
biomarkers of DNA damage.
BACKGROUND Although it has recognized that ischemia-reperfusion can induce oxidative DNA damage, its
occurrence in patients undergoing PTCA has not yet been demonstrated.
METHODS
Three groups of patients were enrolled: 30 patients with documented coronary heart disease
who underwent elective PTCA (group I); 25 patients who underwent elective coronary
angiography for diagnostic purpose (group II); and 27 healthy, age- and gender-matched subjects
(group III). For each subject, the frequency of micronucleated binucleated (MNBN) cells, DNA
single-strand breaks (SSBs), endonuclease III-sensitive sites, and sites sensitive to formamidopyrimidine glycosylase (FPG) were analyzed before and after diagnostic procedures.
RESULTS
The mean basal values of MNBN cells (p ⫽ 0.04), DNA-SSBs (p ⫽ 0.001), endonuclease
III-sensitive sites (p ⫽ 0.002), and FPG sites (p ⬍ 0.0001) were significantly higher in groups
I and II than in group III. A high significant increase of MNBN cell frequency was observed
in group I after the PTCA procedure (11.0 ⫾ 1.3 vs. 19.8 ⫾ 1.6, p ⬍ 0.0001), whereas no
significant difference was observed in group II (10.2 ⫾ 1.3 vs. 12.9 ⫾ 1.4, p ⫽ 0.18). A
significant positive correlation was observed between the increase in the MNBN cell rate and
total inflation time during PTCA (R ⫽ 0.549, p ⫽ 0.0017). The levels of DNA-SSBs (11.7
⫾ 1.4 vs. 26.5 ⫾ 3.0, p ⫽ 0.0003) and FPG sites (13.8 ⫾ 1.8 vs. 22.5 ⫾ 2.4, p ⫽ 0.01) were
also higher after PTCA.
CONCLUSIONS Our results provide evidence for oxidative DNA damage after PTCA, likely related to
ischemia-reperfusion injury. (J Am Coll Cardiol 2002;40:862– 8) © 2002 by the American
College of Cardiology Foundation
OBJECTIVES
Reactive oxygen species, such as superoxide anion, hydrogen
peroxide, peroxynitrite, and hydroxyl radical, seem to play a
pivotal role in the pathogenesis of ischemia-reperfusion
injury (1). Free radicals exert their cytotoxic effects by
producing several metabolic dysfunctions, including the
peroxidation of polyunsaturated fatty acids in membrane or
plasma lipoproteins, direct inhibition of mitochondrial respiratory chain enzymes, inactivation of membrane sodium
channels, and other protein modifications (2,3). Moreover,
it is well known that deoxyribonucleic acid (DNA) damage
frequently occurs in cells exposed to oxidative stress (4).
The presence of free radicals has been demonstrated after
percutaneous transluminal coronary angioplasty (PTCA)
(5– 8), but at the present time, the induction of DNA
damage by free radicals in the circulating cells of patients
undergoing PTCA has not yet been demonstrated.
Therefore, this study was designed to investigate, by
using the micronucleus test and comet assay in the human
lymphocytes of patients undergoing PTCA, whether this
procedure, considered to be a model of ischemia-reperfusion
injury, may cause genetic damage.
We have to consider that the induction of micronuclei in
circulating human lymphocytes is a well-known cytogenetic
test widely employed to assess the genetic effects of spontaneously and mutagenically induced DNA damage (9).
The comet assay is considered a sensitive marker of
oxidative stress and DNA damage (10). Indeed, the use of
lesion-specific enzymes, such as endonuclease III and formamidopyrimidine glycosylase (FPG), which create DNA
strand breaks at sites of oxidative damage, is a fast approach
for measuring of oxidative DNA damage (11,12).
Our study was performed in a group of patients who
underwent elective PTCA for the presence of coronary
stenosis. Moreover, to exclude a hypothetical direct effect of
X-ray exposure and contrast media on DNA damage, a
second group of patients who underwent diagnostic coronary angiography for the presence of myocardial ischemia
on effort was also included.
From the Laboratory of Cellular Biology, CNR Institute of Clinical Physiology, G.
Pasquinucci Hospital, Massa, Italy. This study was entirely supported by grants from
the Italian National Research Council.
Manuscript received March 26, 2001; revised manuscript received May 2, 2002,
accepted May 24, 2002.
METHODS
Patients. Two groups of patients characterized by the
documentation of myocardial ischemia on a stress echocar-
Andreassi et al.
Oxidative DNA Damage and PTCA
JACC Vol. 40, No. 5, 2002
September 4, 2002:862–8
Abbreviations and Acronyms
CAD ⫽ coronary artery disease
DNA ⫽ deoxyribonucleic acid
FPG
⫽ formamidopyrimidine glycosylase
MNBN ⫽ micronucleated binucleated
PTCA ⫽ percutaneous transluminal coronary
angioplasty
SSB
⫽ single-strand breaks
diogram with dipyridamole, performed according to the
standard protocol of use in our institute (13), were consecutively recruited: 30 patients (25 men and 5 women; mean
age 61.3 ⫾ 2.0 years) undergoing elective PTCA (group I)
and 25 patients (19 men and 6 women; mean age 64.4 ⫾ 2.5
years) undergoing diagnostic coronary angiography (group
II). In addition, 27 healthy subjects (19 men and 8 women;
mean age 60.5 ⫾ 1.4 years) matched for age and gender
with the patients of groups I and II were also included as
control subjects (group III).
The severity of coronary artery disease (CAD) was
expressed simply by the number of affected vessels (one-,
two-, or three-vessel disease). Moreover, the extent and
severity of CAD were also evaluated by using the Duke
scoring system (14), with a grade scale ranging from 0 to
100 (0 ⫽ no disease, 100 ⫽ most severe disease).
Patients with episodes of angina within the last 24 h
before the procedure and myocardial infarction within the
last three months were excluded. All patients in whom the
procedure was performed on an urgency or emergency basis
were also excluded.
For the procedures, the patients received nonionic contrast media (Ipromid, Ultravist 370, Schering, Germany).
All patients gave written, informed consent to be in-
cluded in the present study, which was approved by our local
ethical committee.
Venous blood samples were obtained the day before and
three days after the clinical and interventional procedures.
This interval between the two samples was chosen to
perform the micronucleus test and comet assay in peripheral
lymphocytes after a relatively extended period of time from
the procedure.
The demographic and clinical data, angiographic findings, and procedural variables of the study patients are
reported in Table 1.
Cytokinesis block assay and lymphocyte cultures. This
technique employs cytochalasin B to interrupt the division
of each cell before cytokinesis. Therefore, cells that have
completed only one nuclear division can be recognized by
their binucleated appearance (9).
The micronucleus is a small chromatinic particle visible in
interphasic cells close to the main nucleus. It can come from
an acentric chromosomal fragment or from a whole chromosome not correctly incorporated in one of the two
daughter cells at the end of the mitotic division. Thus,
micronuclei are a reliable biomarker of exposure to both
clastogen and aneuploidogen agents that affect the spindle
apparatus.
Two separate cultures from each sample were set up by
mixing 0.3 ml of whole blood with 4.7 ml RPMI 1640
medium (ICN Pharmaceuticals Inc., Irvine, California),
supplemented with 10% fetal calf serum (ICN), 1.5%
phytohemagglutinin (Wellcome, Uxbridge, UK), and antibiotics (100 IU/ml penicillin and 100 mg/ml streptomycin;
Sigma, St. Louis, Missouri). Cultures were maintained for
72 h at 37°C in a humidified atmosphere of 5% carbon
dioxide in air, and cells were blocked in cytokinesis by
adding 6 ␮g/ml cytochalasin B (Sigma) after 44-h incuba-
Table 1. Demographic and Clinical Characteristics
Age (yrs)
Males
Smokers
Duke score
Ejection fraction (%)
Risk factors
Hypertension
Diabetes
Dyslipidemia
Medications
Oral nitrate
Calcium antagonist
Beta-blocker
Aspirin
Procedural variables
Total inflation time (s)
Fluoroscopy exposure rate (Gy/cm2)
Fluoroscopy exposure time (min)
863
Group I
(n ⴝ 30)
Group II
(n ⴝ 25)
Group III
(n ⴝ 27)
61.3 ⫾ 2.0
25
21
36.9 ⫾ 1.7
53.7 ⫾ 1.4
64.4 ⫾ 2.5
19
13
30.5 ⫾ 6.1
49.9 ⫾ 2.5
60.5 ⫾ 1.4
19
10
16
7
22
15
5
15
NS
NS
NS
21
16
12
17
12
8
10
9
NS
NS
NS
NS
115.6 ⫾ 12.0
144.1 ⫾ 18.1
18.3 ⫾ 1.9
—
55.5 ⫾ 8.7
9.1 ⫾ 1.2
0.0001
0.0003
Data are presented as the mean value ⫾ SEM or number of patients or control subjects.
NS ⫽ not significant.
p Value
NS
NS
0.04
NS
NS
864
Andreassi et al.
Oxidative DNA Damage and PTCA
tion. Harvesting of cells, hypotonic treatment, fixation, and
slide preparation were performed according to the standard
method of use in our laboratory (15). For each sample,
1,000 binucleated cells were scored blindly under an optical
microscope (final magnification ⫻400) for analysis of micronuclei, following the criteria for acceptance of micronuclei (9). We evaluated the micronucleated binucleated
(MNBN) cell frequency as the number of micronucleated
cells per 1,000 cells.
Comet assay. PRIMARY DNA DAMAGE. The alkaline
single-cell gel-electrophoresis assay was performed in 10
patients in group I, 10 patients in group II, and 11 subjects
in group III (healthy control subjects). In its alkaline
version, the formation of DNA single-strand breaks (SSBs)
becomes apparent, and the amount of DNA migration, a
“comet-like tail,” indicates the amount of DNA damage in
the cell.
The assay was performed according to the method
previously described (10 –12). Briefly, isolated leukocytes
were suspended in a solution of 0.5% low-melting agarose
and pipetted on a microscope slide precoated with 1%
normal-melting agarose. Then, the slides were immersed in
a lysis solution containing Triton X-100 and 2.5 mol/l NaCl
to lyse the cells and allow DNA unfolding. The resulting
nucleoids were electrophoresed for 25 min at 25 V (300
mA) in alkaline buffer (1 mmol/l Na2EDTA, 300 mmol/l
NaOH, pH ⬎13).
OXIDATIVE DNA DAMAGE. The measurement of oxidative
DNA damage was performed by using specific repair
enzymes that induce breaks in the DNA sites of oxidative
damage. Endonuclease III was used to detect oxidized
pyrimidines, and formamidopyrimidine glycosylase (FPG)
was used to detect damaged purines, including 8-oxoguanine (11,12). After lysis, the slides were incubated at
37°C for 45 min with endonuclease III and 30 min with
FPG. Then, the slides were washed in a neutralizing buffer
(0.4 mol/l Tris-HCl, pH 7.5) and stained with 50 ␮g/ml
ethidium bromide and covered with a cover slip.
For each subject, 50 comets were analyzed under a
fluorescence microscope. The percentage of DNA in the tail
was taken as a measure of DNA break frequency. The net
amount of damage represented by endonuclease IIIsensitive or FPG-sensitive sites was obtained by subtracting
the percent DNA in the tail without enzyme (i.e., strand
breaks) from the percent DNA in the tail with enzyme
incubation.
Comets in each gel were analyzed in a blinded manner,
using the Komet 3.0 image program (Kinetic Imaging Ltd.,
Liverpool, U.K.).
Statistics. The results are expressed as the mean value ⫾
SEM. The data for the three independent groups (I, II, and
III) were analyzed by analysis of variance, and significant
differences between the pairs of mean values were tested by
the Scheffé test. The Scheffé test was chosen for multiple
comparisons because it is very impermeable to violations of
the assumptions typically associated with the multiple com-
JACC Vol. 40, No. 5, 2002
September 4, 2002:862–8
parisons procedure. Statistical evaluations of differences
before and after PTCA were performed by using the paired
Student t test. The association between an increase in DNA
and the effect of each procedural variable (e.g., total inflation
time needed for PTCA, radiation dose, fluoroscopy exposure time) was evaluated by simple regression analysis.
Multiple regression analysis was also applied to investigate
the effect of each variable (e.g., age, gender, smoking status,
atherogenic risk factors, total inflation time, radiation dose,
fluoroscopy exposure time) in determining the MNBN cell
increase. Statistical analyses were performed using the
statistical package Statview, version 5.0 (SAS Institute,
Abacus Concepts, Inc., Berkeley, California).
RESULTS
Relationship between oxidative DNA damage and CAD.
Typical examples of DNA alterations observed in patients
are reported in Figure 1.
When analyzing the DNA damage by the micronucleus
test, the number of basal MNBN cells was higher in groups
I and II than in group III (11.0 ⫾ 1.3 and 10.2 ⫾ 1.3 vs. 7.0
⫾ 0.9, p ⫽ 0.04).
The mean values of DNA-SSBs (p ⫽ 0.001), endonuclease III-sensitive sites (p ⫽ 0.002), and FPG sites (p ⬍
0.0001) were significantly higher in groups I and II (Fig. 2).
Moreover, the mean of FPG-sensitive sites increased
with the number of affected vessels (17.2 ⫾ 2.9, 13.9 ⫾ 2.4,
and 35.8 ⫾ 0.5 for one-, two-, and three-vessel disease,
respectively; p ⫽ 0.004). A significant relationship was
found between FPG-sensitive sites and the extent of CAD
evaluated by means of the Duke scoring system (r ⫽ 0.663,
p ⫽ 0.01).
DNA damage and PTCA. Significant differences were
observed in the radiation dose (144.1 ⫾ 18.1 vs. 55.5 ⫾ 8.7
Gy/cm2, p ⫽ 0.0001) and fluoroscopy exposure time (18.3 ⫾
1.9 vs. 9.1 ⫾ 1.2 min, p ⫽ 0.0003) between groups I and II.
A high significant MNBN cell frequency increase was
observed in group I after PTCA (11.0 ⫾ 1.3 vs. 19.8 ⫾ 1.6,
p ⬍ 0.0001), whereas no significant difference was observed
in group II after coronary angiography (10.2 ⫾ 1.3 vs. 12.9
⫾ 1.4, p ⫽ NS) (Fig. 3). A significant positive correlation
was observed between the increase in the MNBN cell rate
and the total inflation time during PTCA (r ⫽ 0.549, p ⫽
0.0017) (Fig. 4), although no significant relationships were
found between the MNBN cell increase and radiation dose
or fluoroscopy exposure time. Finally, multiple regression
analysis showed that the total inflation time appeared to be
the only determining factor in the increase of MNBN cell
frequency (p ⫽ 0.02).
Oxidative DNA damage and PTCA. The mean levels of
DNA strand breaks, measured as the percentage of DNA in
the tail, were significantly higher in group I after PTCA
(11.7 ⫾ 1.4 vs. 26.5 ⫾ 3.0, p ⫽ 0.0003), although no
significant difference was observed in group II after coronary
angiography (12.5 ⫾ 2.8 vs. 11.6 ⫾ 1.5, p ⫽ NS) (Fig. 5).
JACC Vol. 40, No. 5, 2002
September 4, 2002:862–8
Andreassi et al.
Oxidative DNA Damage and PTCA
865
Figure 1. An example of micronucleated binucleated cells with two micronuclei (A) and cell migration patterns produced by the comet assay (B).
Moreover, the FPG-sensitive sites were strongly increased
after PTCA (13.8 ⫾ 1.8 vs. 22.5 ⫾ 2.4, p ⫽ 0.01), and a
trend toward an increase, although not significant, was
observed for the levels of endonuclease III (18.7 ⫾ 3.4 vs.
27.1 ⫾ 3.1, p ⫽ 0.08). No significant differences were
observed in the levels of FPG-sensitive sites and endonuclease III-sensitive sites in group II after coronary angiography.
DISCUSSION
This study shows that patients with CAD, in the basal
condition, have a higher level of DNA damage, as compared
with normal subjects, according to our previous observation
(15). Moreover, to the best of our knowledge, this study
provides the first evidence of possible oxidative DNA
damage in the peripheral lymphocytes of patients undergoing PTCA.
Comparison with other studies. A number of previous
studies have shown an increase in cardiac oxidative stress
induced by an injury incurred during primary angioplasty
(5– 8). Recently, it has also been demonstrated that lipid
peroxidation and F2-isoprostane—stable end products of
oxygen free radical–mediated lipid peroxidation—are signif-
icantly increased during the reperfusion after PTCA (8,16).
Although DNA represents a potential target for free radical
attack, no evidence is yet available demonstrating DNA
damage as a result of ischemia-reperfusion injury after
PTCA.
Indeed, it has been shown that oxidation is a major cause
of DNA damage in humans, and the number of oxidative
hits on DNA may be ⬎103/cell per day (17). Reactive
oxygen species cause extensive DNA damage, including
SSBs, the formation of modified bases, chromosomal damage, and mutations in mammalian cells (4,18). Several
experimental models of ischemic injury have also shown the
influence of reperfusion-associated oxygen radicals on DNA
damage. In fact, some studies have shown that ischemiareperfusion induces DNA damage in animal studies and
isolated tissue (19 –21).
Recent studies have reported that the induction of apoptosis is an initial cellular event induced by balloon angioplasty (22,23). Indeed, it is well known that apoptosis is an
effective mechanism for eliminating cells with genetic lesions, thus reducing the potential for genetic instability and
cellular dysfunction (24). In addition, the induction of
chromosomal and DNA damage has been demonstrated in
866
Andreassi et al.
Oxidative DNA Damage and PTCA
JACC Vol. 40, No. 5, 2002
September 4, 2002:862–8
Figure 3. Frequency of micronucleated binucleated cells in patients undergoing percutaneous transluminal coronary angiography (group I) and
coronary angiography (group II). Open bars ⫽ before procedure; solid
bars ⫽ after procedure.
Figure 2. Deoxyribonucleic acid (DNA) damage, as demonstrated by
single strand breaks (SSBs), formamidopyrimidine glycosylase (FPG) sites,
and endonuclease III (E III) sites, in the lymphocytes of patients classified
into three study groups, represented by the mean percent DNA in the tail
for 50 comets from each patient.
human peripheral leukocytes after ischemia-reperfusion injury (25,26).
Value and limitations of the study. Our study demonstrates the presence of DNA damage in the lymphocytes of
patients with CAD, who represent an ideal situation of
increased production of oxygen free radicals. These results
are in agreement with another study reporting higher DNA
adduct levels in the cardiac tissue of patients with severe
CAD (27), as well as with other recent observations
suggesting that alterations at the DNA level may contribute
significantly to the development of atherosclerotic disease
(28 –30).
Our results show that PTCA induces an increase in
oxidative DNA damage in the circulating cells of patients.
These effects were seen in human lymphocytes of the
systemic circulation. Peripheral lymphocytes are advanta-
geous for exposure analysis because they circulate throughout the body and thus provide an estimate of average
whole-body exposure. In fact, the conceptual basis for this
approach is the hypothesis that the extent of genetic damage
in peripheral blood lymphocytes reflects critical events for
mutagenic and carcinogenic processes in target tissues.
Similarly, specific genetic changes may occur in smooth
muscle cells of the wall, leading to restenosis, as suggested
by an increased mutation rate of microsatellites in both
primary and secondary restenotic atherosclerotic plaques
(31). It is also possible that the observed DNA damage may
increase apoptosis (22,23). However, a recent study suggested that restenotic intimal cells may be resistant to
apoptosis (32), and increased DNA synthesis has been
observed after balloon angioplasty in an atherosclerotic
rabbit model (33). This evidence suggests that intimal
hyperplasia of smooth muscle cells may be a result of their
clonal growth in response to a mutagenic event, underscor-
Figure 4. Relationship between micronucleated binucleated (MNBN) cell
increase after percutaneous transluminal coronary angiography and total
inflation time. y ⫽ 1.123 ⫹ 0.072x; r ⫽ 0.549, p ⫽ 0.002.
Andreassi et al.
Oxidative DNA Damage and PTCA
JACC Vol. 40, No. 5, 2002
September 4, 2002:862–8
867
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Figure 5. Deoxyribonucleic acid (DNA)-single strand breaks (SSBs) in
patients undergoing percutaneous transluminal coronary angiography
(group I) and coronary angiography (group II). Open bars ⫽ before
procedure; solid bars ⫽ after procedure.
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The hypothesis that oxygen free radicals may be responsible for these alterations is plausible (4,34,35); however,
some limitations of this study should also be considered.
First and foremost, the possibility that X-ray exposure and
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additional prognostic factor in CAD, as well as to investigate the association between oxidative DNA damage and
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Acknowledgments
We acknowledge the help given by the paramedical and
technical staff of our hemodynamic laboratory.
Reprint requests and correspondence: Dr. Maria Grazia Andreassi, CNR Institute of Clinical Physiology, G. Pasquinucci Hospital, Via Aurelia Sud-Montepepe 54100, Massa, Italy. E-mail:
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